The use of prostate specific antigen (PSA) as a diagnostic biomarker for prostate cancer was approved by the US Federal Drug Agency in 1994. In the nearly two decades since this approval, the PSA test has remained the primary tool for use in prostate cancer diagnosis, in monitoring for recurrence of prostate cancer, and in following the efficacy of treatments. However the PSA test has multiple shortcomings and, despite its widespread use, has resulted in only small changes in the death rate from advanced prostate cancers. To reduce the death rate and the negative impacts on quality of life caused by prostate cancer, new diagnostic tools are required not only for more accurate primary diagnosis, but also for assessing the risk of spread of primary prostate cancers, and for monitoring responses to therapeutic interventions.
Today, a blood serum level of around 4 ng per ml of PSA is considered indicative of prostate cancer, while a PSA level of 10 ng per ml or higher is considered highly suggestive of prostate cancer. The PSA blood test is not used in isolation when checking for prostate cancer; a digital rectal examination (DRE) is usually also performed. If the results of the PSA test or the DRE are abnormal, a biopsy is generally performed in which small samples of tissue are removed from the prostate and examined. If the results are positive for prostate cancer, further tests may be needed to determine the stage of progression of the cancer, such as a bone scan, a computed tomography (CT) scan or a pelvic lymph node dissection.
While the PSA test has a good sensitivity (80%), it suffers from a false positive rate that approaches 75% due mainly to the presence of benign prostatic hyperplasia (BPH), another prostatic condition that results in elevated PSA levels. For example, it has been estimated that for PSA values of 4-10 ng/ml, only one true diagnosis of prostate cancer was found in approximately four biopsies performed (Catalona et al. (1994) J. Urol. 151(5):1283-90). Tests that measure the ratio of free to total (i.e., free plus bound) PSA do not have significantly greater specificity or sensitivity than the standard PSA test.
Higher PSA levels often lead to biopsies to determine the presence or absence of cancer cells in the prostate, and may lead to the surgical removal of the localized prostate gland. While surgery removes the localized cancer and often improves prostate cancer-specific mortality, it also masks the fact that many patients with prostate cancer, even in the absence of surgery, do not experience disease progression to metastasis or death.
Currently, the established prognostic factors of histological grade and cancer stage from biopsy results, and prostate-specific antigen level in blood at diagnosis are insufficient to separate prostate cancer patients who are at high risk for cancer progression from those who are likely to die of another cause.
Once virulent forms of prostate cancer have been diagnosed, control strategies may involve surgery to remove the prostate gland if identified before metastasis, radiation to destroy cancer cells within the prostate, and drug-based testosterone repression, generally referred to as androgen depletion therapy. These various treatments may bring about cures in some instances, or slow the time to death. However, for those with the most virulent forms of prostate cancer, the cancer will usually recur after surgery or radiation therapy and progress to resistance to androgen depletion therapy, with death a frequent outcome.
Early detection of virulent forms of prostate cancer is critical but the conclusion of specialist physicians is that the PSA test alone is inadequate for distinguishing patients whose cancers will become virulent and progress to threaten life expectancy from those with indolent cancers.
The following are some key reasons why the PSA test does not meet the needs of men's health:
i) The Type of Cancer
There are at least two basic cell types involved in prostate cancer. Adenocarcinoma is a cancer of epithelial cells in the prostate gland and accounts for approximately 95% of prostate cancers. Neuroendocrine cancers may arise from cells of the endocrine (hormonal) and nervous systems of the prostate gland and account for approximately 5% of prostate cancers. Neuroendocrine cells have common features such as special secretory granules, produce biogenic amines and polypeptide hormones, and are most common in the intestine, lung, salivary gland, pituitary gland, pancreas, liver, breast and prostate. Neuroendocrine cells co-proliferate with malignant adenocarcinomas and secrete factors which appear to stimulate adenocarcinoma cell growth. Neuroendocrine cancers are rarer, and are considered non-PSA secreting and androgen-independent for their growth.
ii) Asymptomatic Men
Some 15 to 17% of men with prostate cancer have cancers that grow but do not produce increasing or high blood levels of PSA. In these patients, who are termed asymptomatic, the PSA test often returns false negative test results as the cancer grows.
iii) BPH, Prostatitis and PIN
Benign prostate hypertrophy (BPH), a non-malignant growth of epithelial cells, and prostatitis are diseases of the prostate that are usually caused by an infection of the prostate gland. Both BPH and prostatitis are common in men over 50 and can result in increased PSA levels. Incidence rates for BPH increase from 3 cases per 1000 man-years at age 45-49 years, to 38 cases per 1000 man-years by the age of 75-79 years. Whereas the prevalence rate of BPH is 2.7% for men aged 45-49, it increases to at least 24% by the age of 80 years. While prostate cancer results from the deregulated proliferation of epithelial cells, BPH commonly results from proliferation of normal epithelial cells and frequently does not lead to malignancy (Ziada et al. (1999) Urology 53(3 Suppl 3D):1-6). Prostatitis in males is frequently attributed to an infection in prostate tissue including infection by E. coli, Klebsiella sp., Serratia and/or Pseudomonas sp.
Another condition, known as prostate intraepithelial neoplasia (PIN), may precede prostate cancer by five to ten years. Currently there are no specific diagnostic tests for PIN, although the ability to detect and monitor this potentially pre-cancerous condition would contribute to early detection and enhanced survival rates for prostate cancer.
iv) The Phenotype of the Prostate Cancer
The phenotype of prostate cancer varies from one patient to another. More specifically, in different individuals prostate cancers display heterogeneous cellular morphologies, growth rates, responsiveness to androgens and pharmacological blocking agents for androgens, and varying metastatic potential. Each prostate cancer has its own unique progression involving multiple steps, including progression from localized carcinoma to invasive carcinoma to metastasis. The progression of prostate cancer likely proceeds, as seen for other cancers, via events that include the loss of function of cell regulators such as cancer suppressors, cell cycle and apoptosis regulators, proteins involved in metabolism and stress response, and metastasis related molecules (Abate-Shen et al. (2000) Polypeptides Dev. 14(19):2410-34; Ciocca et al. (2005) Cell Stress Chaperones 10(2):86-103).
There is unlikely to be one diagnostic test that detects all forms of primary prostate cancer, or one prognostic test for all routes to metastasis. Further tests are therefore needed before prostate cancer can be diagnosed in its different forms.
At present health authorities, do not universally recommend widespread screening for prostate cancer with the PSA test. There are concerns that many men may be diagnosed and treated unnecessarily as a result of being screened, at high cost to health systems as well as risking the patient's quality of life, such as through incontinence or impotence. Despite these concerns, prostate cancer is the most prevalent form of cancer and the second most common cause of cancer death in New Zealand, Australian and North American males (Jemal et al. (2007) CA Cancer J. Clin. 57(1):43-66). In reality, many men incubating life threatening forms of prostate cancer are being missed until their cancer is well advanced, due to the economic costs of national screening, the need to avoid unnecessary over-treatment, and/or the presence of progressive cancers producing only low or background levels of PSA. The need for a better diagnostic test could not be clearer.
An important clinical question is how aggressively to treat patients with localized prostate cancer. Treatment options for more aggressive cancers are invasive and include radical prostatectomy and/or radiation therapy. Androgen-depletion therapy, for example using gonadotropin-releasing hormone agonists (e.g., leuprolide, goserelin, etc.), is designed to reduce the amount of testosterone that enters the prostate gland and is used in patients with metastatic disease, some patients who have a rising PSA and choose not to have surgery or radiation, and some patients with a rising PSA after surgery or radiation. Treatment options usually depend on the stage of the prostate cancer. Men with a 10-year life expectancy or less, who have a low Gleason score from a biopsy and whose cancer has not spread beyond the prostate are often not treated. Younger men with a low Gleason score and a prostate-restricted cancer may enter a phase of “watchful waiting” in which treatment is withheld until signs of progression are identified. However, these prognostic indicators do not accurately predict clinical outcome for individual patients.
Unlike many cancer types, specific patterns of oncogene expression have not been consistently identified in prostate cancer progression, although a number of candidate genes and pathways likely to be important in individual cases have been identified (Tomlins et al., Annu. Rev. Pathol. 1:243-71, 2006). Several groups have attempted to examine prostate cancer progression by comparing gene expression of primary carcinomas to normal prostate. Because of differences in technique, as well as the true biologic heterogeneity seen in prostate cancer, these studies have reported thousands of candidate genes but shared only moderate consensus.
A few genes have emerged including hepsin (HPN; Rhodes et al., Cancer Res. 62:4427-33, 2002), alpha-methylacyl-CoA racemase (AMACR; Rubin et al., JAMA 287:1662-70, 2002) and enhancer of Zeste homolog 2 (EZH2; Varambally et al., Nature 419:624-9, 2002), which have been shown experimentally to have probable roles in prostate carcinogenesis. Most recently, bioinformatic approaches and gene expression methods have been used to identify fusion of androgen-regulated genes including transmembrane protease, serine 2 (TMPRSS2) with members of the erythroblast transformation specific (ETS) DNA transcription factor family (Tomlins et al., Science 310:644-8, 2005). These fusions appear commonly in prostate cancers and have been shown to be prevalent in more aggressive cancers (Attard et al., Oncogene 27:253-63, 2008; Demichelis et al., Oncogene 26:4596-9, 2007; Nam et al., Br. J. Cancer 97:1690-5, 2007). A number of studies have shown distinct classes of cancers separable by their gene expression profiles (Rhodes et al., Cancer Res. 62:4427-33, 2002; Glinsky et al., J. Clin. Invest. 113:913-23, 2004; Lapointe et al., Proc. Natl. Acad. Sci. USA 101:811-6, 2004; Singh et al., Cancer Cell 1:203-9, 2002; Yu et al., J. Clin. Oncol. 22:2790-9, 2004). In addition, a number of gene expression studies have been performed looking for gene dysregulation in metastatic prostate cancers when compared to normal (healthy) prostate tissue (Varambally et al., Nature 419:624-9, 2002; Lapointe et al., Proc. Natl. Acad. Sci. USA 101:811-6, 2004; LaTulippe et al., Cancer Res. 62:4499-506, 2002). One factor impacting clinical utility of gene expression analyses is the fact that most samples available for validation exist only as formalin fixed paraffin embedded (FFPE) tissues. In contrast, many of the cDNA microarray studies have used snap frozen tissues (Bibikova et al., Genomics 89:666-72, 2007; van't Veer et al., Nature 415:530-6, 2002).
There are a range of blood proteins whose levels change in subjects as prostate cancer develops. Some of these are produced by prostate cancer cells themselves, while others are produced by cells of non-prostate origin.
PSA is a class of protein that is produced by both healthy and cancerous prostate tissues. Another class of proteins that change as prostate cancer develops are immune system products. These can be divided into immune specific products, such as antibodies, and immune nonspecific products, such as growth factors and acute phase proteins.
Acute-phase proteins are a class of proteins whose plasma concentrations increase in response to inflammation. In response to injury and cancer cell growth, local inflammatory cells (neutrophils, granulocytes and macrophages) secrete a number of cytokines into the bloodstream, most notable of which are the interleukins IL-1, IL-6 and IL-8, and TNF-α. As a result, acute phase proteins such as C-reactive protein (CRP) may be produced in the liver and secreted into the blood. CRPs physiological role is to bind to phosphocholine expressed on the surface of dead or dying cells (and some types of bacteria) in order to activate the complement system via the C1q complex. Serum amyloid A proteins are another class of acute phase proteins and are a family of apolipoproteins associated with high-density lipoprotein (HDL) in plasma that increase in levels in response to inflammatory stimuli. Cysteine-rich secretory protein 3 (CRISP3) is another acute-phase protein, expressed mainly in mononuclear cells. Its expression is reportedly upregulated in prostate cancer, and is a precursor of recurrence after radical prostatectomy for localized prostate cancer.
Anti-nuclear antibodies (ANA) are autoantibodies directed against the contents of the cell nucleus. They are present in higher than normal numbers in autoimmune disease. The ANA test measures the pattern and amount of autoantibody which can attack the body's tissues as if they were foreign material. Autoantibodies are present in low titers in the general population, but in about 5% of the population their concentration is increased, and about half of this 5% have an autoimmune disease.
Some antigens are found predominantly associated with cancer cells and are termed cancer antigens. CA-125 (cancer antigen 125 or carbohydrate antigen 125; also known as mucin 16 or MUC16) is a member of the mucin family of glycoproteins which may be elevated in the blood of some patients with pancreatic or breast cancer. Carcinoembryonic antigen (CEA) is a type of protein molecule that can be found in many different cells of the body, but is typically associated with certain cancers and the developing fetus. The word “carcinoembryonic” reflects the fact that CEA is produced by some cancers and by the developing fetus.
Typical over-reactions of the host immune response and delayed hypersensitivity reactions are represented by inflammatory infiltrates from T-lymphocytes (CD4+ T helper/inducer cells and CD8+ T cytotoxic/suppressor cells), which are distributed variously between the epithelial and stromal components. Some men with symptoms of chronic prostatitis have evidence of a proliferative CD4/T-cell response to PSA. T-lymphocytes secrete inflammatory mediators such as the complement components C3, C4 and IL-6 in the serum. The concentrations of these markers decrease with the relief of prostatitis symptoms.
The concept of using more than one biomarker to diagnose prostate cancer to improve the accuracy of the PSA test and to increase its prognostic value has been analyzed by a number of investigators. Patterns of biomarker levels in serum have been compared to cancer staging tools as the T (type) classification, N (node) classification, Gleason score and prostate specific antigen (PSA) value before therapy and disease grade. Serum levels of alkaline phosphatase (ALP), lactate dehydrogenase (LDH), chromogranin A (CHGA), serum calcium, and hemoglobin platelet count indicators that add to the accuracy of a PSA value are used by physicians to assess treatment modalities but have not been widely used for prognostic assessment. Large retrospective clinical studies have been undertaken to determine whether disease-specific survival assessed using univariate and multivariate Cox's proportional hazards model analyzes is correlated with patterns of biomarker expression before, during and after treatment.
New prognostic indices clinically applicable for patients with metastatic or late stage (Stage IV) prostate cancer are needed to assess efficacy of treatments because prostate-specific antigen (PSA) tests fail to reflect the prognostic outcome. The PSA/prostatic acid phosphatase (PAP) ratio has been tested as a prognostic index while serum levels of CRP, PSA, CHGA and ALP have been shown to increase and haemoglobin levels have been shown to decrease with advancing extent of disease (EOD) on bone scan EOD grade, and have variously been associated with disease-specific survival. However none of these biomarkers have entered routine diagnostic or prognostic use for prostate cancer.
There thus remains a need in the art for an accurate test for all forms of prostate cancer.